Method of stabilizing a bioactive peptide against protease hydrolysis

ABSTRACT

The invention includes a thioamide-modified peptide, wherein the thioamide modification increases the in vivo half-life of the peptide. The invention further includes methods of treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a thioamide-modified peptide of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 15/305,681, filed Oct. 21, 2016, nowallowed, which is a 35 U.S.C. § 371 national phase application from, andclaims priority to, International Application No. PCT/US2015/028008,filed Apr. 28, 2015, and published under PCT Article 21(2) in English,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalApplication No. 61/985,045, filed Apr. 28, 2014, all of whichapplications are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 4, 2018, isnamed 046483-7016US2_Sequence_Listing.txt and is 28.3 kilobytes in size.

BACKGROUND OF THE INVENTION

In recent years, there has been significant interest in the developmentof biologic drugs, including peptide biologics (Multard, 2013, Nat. Rev.Drug Discovery 12:329-332; Projan, et al., 2004, Expert Opin. Biol.Therapy 4:1345-1350; Verdine, et al., 2007, Clin. Cancer Res.13:7264-7270). Peptide biologics have distinct advantages over smallmolecule drugs. Biologics are typically based on natural bioactivepeptides (such as hormones and neuropeptides), and this makes theidentification of a “lead compound” much easier than the identificationof a small molecule lead compound (Buse, et al., 2009, Lancet 374:39-47;Kreymann, et al., 1987, Lancet 330:1300-1304).

Unfortunately, peptides generally make poor drugs because they undergorapid proteolysis in vivo, leading to unfavorable pharmacokinetics(Weber, 2004, J. Med. Chern. 47:4135-4141). As a consequence, much ofthe time in development of peptide biologics is spent on modifying thepeptide to reduce proteolysis, while maintaining biological activity(Buse, et al., 2009, Lancet 374:39-47; DeFronzo, et al., 2005, DiabetesCare 28:1092-1100). Some of the strategies reported in the art make useof the incorporation of unnatural amino acids (such as D-amino acids orβ-amino acids) into the peptidic chain to overcome proteolysis (Bird, etal., 2010, Proc. Natl. Acad. Sci. 107:14093-14098; Sato, et al., 2006,Curr. Opin. Biotechnol. 27:638-642). Such approaches requireidentification of appropriate modification sites and are generally timeconsuming and often met with failure.

Heart disease is two times more common among obese adults and four timesmore common among diabetic adults, and consistently these conditionsaffect large numbers of U.S. adults, where 35% of adults are obese and27% of those 65 and older have diabetes (90% of diabetes cases are Type2). Both conditions are strongly regulated by peptide hormones and thusamenable to therapeutic intervention. Aside from insulin and glucagon,one of the most well characterized peptides in this class is thegut-derived incretin hormone glucagon-like peptide 1 (GLP-1) (Kreymann,et al., 1987, Lancet 330:1300-1304).

The GLP-1 7-36 residue fragment, which is referred to simply as “GLP-1”hereinafter (SEQ ID NO:1), stimulates insulin and suppresses glucagonsecretion, inhibits gastric emptying, and reduces appetite and foodintake (Drucker, et al., 2006, Lancet 368:1696-1705). Glucose-stimulatedinsulin secretion (GSIS) is a phenomenon wherein certain compounds(natural or synthetic) augment the release of insulin from pancreaticβ-cell islets in the presence of glucose. These reagents have no effecton insulin secretion in the absence of glucose, and display twoadvantages over direct stimulators of insulin secretion. First, sincethey only cause increased insulin secretion in the presence of glucose,they augment the natural physiological mechanism for insulin secretion.Second, compounds that directly stimulate insulin secretion can causeβ-cell stress and lead to the death of these vital cells (Maedler, etal., 2005, J. Clin. Endocrinol. Metab. 90:501-506). However, simpletreatment by GLP-1 injection is not feasible because it is inactivatedthrough proteolytic cleavage by dipeptidyl peptidase 4 (DPP-4) with ahalf-life of less than 2 minutes (Kim, et al., 2008, Pharmacol. Rev.60:470-512). DPP-4 preferentially cleaves after Pro or Ala residuespenultimate to the N-terminus and functions as the principal determinantof the circulating half-life for GLP-1 and many other peptides thataffect cardiac health (Mentlein, et al., 1993, Eur. J. Biochem.214:829-835).

Therapeutic approaches for enhancing incretin action include bothdegradation-resistant GLP-1 receptor (GLP-1R) agonists and inhibitors ofDPP-4 activity. Two stabilized incretin mimetics are currentlyprescribed as injectables taken between once daily and once weekly:exenatide (Byetta®, SEQ ID NO:3) and liraglutide (Victoza®, SEQ IDNO:4). Both induce reductions in fasting, postprandial blood glucoseconcentrations and hemoglobin A1c (1-2%), which is associated withweight loss (2-5 kg). These incretin mimetics also expand pancreaticβ-cell mass, and have emerged, along with DPP-4 inhibitors such assitagliptin (Januvia®), as viable treatments for Type 2 diabetes(Drucker, et al., 2006, Lancet 368:1696-1705). While they act along thesame hormone signaling axis, the two types of therapies are not mutuallyexclusive, as DPP-4 inhibitors fail to produce some desirable effects ofthe peptidomimetics such as appetite suppression and weight loss.Moreover, there is concern that DPP-4 inhibition could increase the riskof cancer (Stulc, et al., 2010, Diabetes Res. Clin. Pract. 88:125-131).DPP-4 exists as both a membrane bound form and a soluble form incirculation due to cleavage of the active site domain from the membrane.Activity of the membrane bound form suppresses non-small cell lungcarcinoma cells (Wesley, et al., 2004, Int. J. Cancer 109:855-866).Given that there are concerns about chronic DPP-4 inhibition and someeffects are unique to stabilized GLP-1 peptides, there is an interest inusing peptides instead of, or in addition to, approved DPP-4 inhibitors.

DPP-4 substrates include not only GLP-1, but also glucose-dependentinsulinotropic factor (GIP; SEQ ID NO:5), oxyntomodulin (OXM; SEQ IDNO:6), and brain natriuretic peptide (BNP; SEQ ID NO:7). All of thesepeptides have half-lives of less than 15 minutes. Similar to GLP-1, thehormones GIP and OXM act as glucose-lowering agents and have beenstudied extensively as diabetes treatments (Meneilly, et al., 1993,Diabetes Care 16:110-114; Cohen, et al., 2003, J. Clin. Endocrinol.Metab. 88:4696-4701). BNP plays an important role in the body's defenseagainst hypertension and is used as a treatment of congestive heartfailure (Del Ry, et al., 2013, Pharmacol. Res. 76:190-198; Grantham, etal., 1997, Am. J. Physiol.-Reg. Int. Comp. Physiol. 272:R1077-R1083).DPP-4 inhibition affects the levels of all of these peptides incirculation. On the other hand, stabilized versions of GIP, OXM, or BNPshould act more selectively than DPP-4 inhibition, by impacting only onesignaling pathway.

A variety of peptidomimetic strategies have already been applied tostabilizing peptide hormones, including GLP-1. Most of the strategiesinvolve restricting DPP-4 access to the cleavable bond. Exenatide doesthis using replacement with other natural amino acids. Liraglutideincludes a fatty acid modified sidechain, which wraps around the peptideand stabilizes a compact conformation. In the known peptides M1 (SEQ IDNO:8; Deacon, et al., 1998, Diabetologia 41:271-278); M2 (SEQ ID NO:9;Heard, et al., 2013, J. Med. Chem. 56:8339-8351) and M3 (SEQ ID NO:10;Iltz, et al., 2006, Clin. Ther. 28:652-665), access to cleavable bondsis blocked and conformations are stabilized with methyl substitutions.These modifications can extend the half-life for DPP-4 proteolysis, butcan compromise GLP-1R affinity (for example, the minimalist M3 has a6-fold lower affinity; Iltz, et al., 2006, Clin. Ther. 28:652-665).Modifications that increase GLP-1 half-life while sacrificing GLP-1Ractivation are less effective at achieving the desired effects ofregulating glucose and promoting weight loss.

There is a need in the art for straightforward methods of stabilizingpeptides, such as peptide hormones or neuropeptides, against proteolyticdegradation in vivo. Such methods should allow for the identification ofa modified peptide with similar potency to, but increased stabilityover, the naturally occurring peptide. The present invention addressesthis unmet need in the art.

BRIEF SUMMARY OF THE INVENTION

The invention relates to unexpected discovery of thioamide-modifiedpeptides that have long half-lives and are resistant to in vivoproteolytic degradation. In one aspect, the invention includes apeptide, or a salt or solvate thereof, comprising at least one selectedfrom the group consisting of SEQ ID NOs:2, 11-18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50-51. In certainembodiments, the C-terminus of the peptide is amide protected.

In yet another aspect, the invention includes a pharmaceuticalcomposition comprising at least one peptide of SEQ ID NOs:2, 11-18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50-51, and apharmaceutically acceptable carrier. The pharmaceutical composition mayfurther comprise at least one additional agent useful for treating orpreventing a disease or disorder in a subject, which in certainembodiments is a human. The additional agent may be co-formulated withthe thioamide-modified peptides. The disease or disorder includes, butis not limited to, diabetes, obesity, hypertension or congestive heartfailure. The composition is administered to the subject by at least oneroute selected from the group consisting of nasal, inhalational,topical, oral, buccal, rectal, pleural, peritoneal, vaginal,intramuscular, subcutaneous, transdermal, epidural, intratracheal, otic,intraocular, intrathecal, and intravenous routes.

In yet another aspect, the invention includes a method of stabilizing apeptide against protease hydrolysis. The method comprises modifying witha thioamide the peptide bond that a protease hydrolyzes. In certainembodiments, the protease comprises DPP-4. In other embodiments, theprotease comprises carboxypeptidase. The peptides that can be modifiedby the method stated herein include, but are not limited to,glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic factor(GIP), oxyntomodulin (OXM), glucagon, pituitary adenylatecyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP),growth-hormone-releasing hormone (GHRH), sermorelin (GRF), peptide YY(PYY), pancreatic polypeptide (PP), B-type natriuretic peptide (BNP),neuropeptide Y (NPY), enterostatin (ENT), RANTES (CCL5), CCL2, CCL8,CCL7, and CCL13. In certain embodiments, the thioamide modification isbetween the second and third amino acid residues from the N-terminus ofthe peptide.

In certain embodiments, the thioamide modification of the peptides ofthe invention retains substantially the same biological activities andstructures of corresponding unmodified peptides, but extends the in vivohalf-lives of unmodified peptides.

In yet another aspect, the invention includes a method of treating orpreventing diabetes or obesity in a subject in need thereof. The methodcomprises administering to the subject a therapeutically effectiveamount of a thioamide-modified peptide. The thioamide-modified peptidecomprises at least one selected from the group consisting of SEQ IDNOs:2, 11-14, 16-18, 20, 22, 48 and 51, or a salt or solvate thereof.The thioamide-modified peptide has at least one effect selected from thegroup consisting of stimulating insulin production in the subject,suppressing glucagon secretion in the subject, inhibiting gastricemptying in the subject, reducing appetite in the subject, and reducingfood intake in a subject.

In yet another aspect, the invention includes a method of treating orpreventing a cardiac disease or disorder in a subject in need thereof.In certain embodiments, the cardiac disease or disorder compriseshypertension or congestive heart failure. The method comprisesadministering to the subject a therapeutically effective amount of athioamide-modified peptide of SEQ ID NO:15, or a salt or solvatethereof.

In yet another aspect, the invention includes a kit comprising athioamide-modified peptide, and an instructional material for usethereof, wherein the instructional material comprises instructions fortreating or preventing a disease or disorder in a subject using thethioamide-modified peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings specific embodiments. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities of the embodiments shown in the drawings.

FIG. 1 illustrates peptide inactivation by DPP-4. Peptides areinactivated by DPP-4-catalyzed cleavage at the bond indicated with aslash. GLP-1 analogs such as exenatide, liraglutide, M1 (taspoglutide),M2, and M3 exhibit increased stability towards DPP-4 degradation.Modified residues relative to GLP-1 are shown in green.

FIG. 2 illustrates thioamide fluorescence quenching applied toproteolysis. Top: Thioamide-containing peptides can be synthesized frombenzotriazole precursors. The thioamide bond is denoted with a primesymbol. Bottom left: Cleavage of the substrate L'LKAAμ by papain led toan increase in fluorescence. No change in fluorescence was observed forthe oxoamide control peptide (LLKAAμ).

FIG. 3 comprises a set of graphs illustrating glucose tolerance tests(GTTs). Left: Short timecourse GTT to measure the activity of GLP-1 (1mg/kg) and exendin-4 (1 mg/kg) in vivo. Right: Comparison of theactivities of GLP-1 (1 mg/kg) and exendin-4 (1 mg/kg) 5.5 hours afterthey were injected showed that the more stable exenatide retainedactivity while GLP-1 did not, due to proteolytic inactivation. Student'st-tests used to determine statistical significance, p-value<0.01, *;p-value<0.0001, ***).

FIG. 4 illustrates thioamide stability assays. Alkyne-modifiedthiopeptides are incubated with cells or lysates and reacted with a“clickable” azido-diazo-biotin (ADB) pulldown reagent to isolate theproducts for HPLC MS analysis.

FIG. 5 comprises a set of graphs illustrating the kinetics of hydrolysisof a thioamide-incorporating peptide using trypsin or chymotrypsin (toppanel) and HPLC analysis of hydrolysis of peptides and correspondingthioamide-incorporating peptides using trypsin (left) or chymotrypsin(right) (bottom panel).

FIG. 6 comprises a set of graphs illustrating thio GLP-1 data. Leftgraph: Comparison of thio GLP-1 and GLP-1 cleavage by DPP-4. Right:Circular dichroism (CD) spectra of 40 μM GLP-1 and thio GLP-1 in bufferwith 30% trifluoroethanol. Thioamide substitution at the GLP-1 scissilebond suppressed proteolysis without disrupting structure (CD) oractivity (in vivo).

FIG. 7 comprises a set of graphs illustrating experimental resultsobtained with thioamide-modified GLP-1 (“thio GLP-1”) as compared withnon-modified GLP-1 (“GLP-1”). The experimental results indicate thatthioamide substituted GLP-1 is active in mice. Intra-peritoneal GlucoseTolerance Test (IPGTT) was used to measure activity of GLP-1 (1 mg/kg)and thio GLP-1 (1 mg/kg) in vivo (Student's t-tests used to determinestatistical significance).

FIG. 8 comprises a set of graphs illustrating the finding thatdegradation of GLP-1-A^(s) ₈ is mitigated compared to that of GLP-1 inpresence of DPP-4. GLP-1-A^(s) ₈ appeared not to bind to DPP-4, and didnot inhibit cleavage of Gly-Pro-pNA substrate at 100 μM:

FIG. 9 illustrates a non-limiting mechanistic pathway of GLP-1-A^(s) ₈auto-degradation. Copper appeared to block auto-degradation.

FIG. 10 comprises a set of graphs illustrating the time-dependentdetection of GLP-1-A^(s) ₈ auto-degradation.

FIG. 11 is a graph illustrating the finding that the thioamidemodification of a peptide (GLP-1-A^(s) ₈) does not significantly alterthe structure of the original peptide (GLP-1).

FIG. 12 is a graph illustrating the finding that GLP-1-A^(s) ₈ activatescAMP production in GT1-7 neurons.

FIG. 13 is a graph illustrating that GLP-1-A^(s) ₈ reduces body massincrease in rats. The rats experienced 15% reduction in food intake over4 hours after i.p. injection. This effect was mitigated by co-injectionof GLP-1-A^(s) ₈ and Exendin-9.

FIG. 14 comprises a set of graphs illustrating that GLP-1-F7A^(s) ₈ isstable in buffer with or without DPP-4. The half-life of GLP-1-F7A^(s) ₈in buffer with or without DPP-4 is about 24 hours. GLP-1-F₇A^(S) ₈ wasnot degraded by DPP-4. EC₅₀s: GLP-1-F7=0.9 nM, GLP-1=0.8 nM.

FIG. 15 illustrates the method of screening thioamide effects inpresence of DPP-4. Short reporter peptides (containing Mcm, μ) allowsfor rapid screening at P1, P2 and P1′ positions.

FIG. 16 illustrates the finding that GLP-1 is degraded by DPP-4, havinga half-life less than 10 minutes.

FIG. 17 illustrates systematic scanning of the effects of thioamidemodification position in a peptide. The thioamide modifications wereconducted at different positions of the peptide chain. The resultingpeptides were mixed with either papain or trypsin enzymes to measure thechanges of fluorescence intensity. The “+” sign after the peptidesequence means the presence of the corresponding enzyme. The “−” signafter the peptide sequence means the lack of the corresponding enzyme.

FIG. 18 comprises a non-limiting scheme to synthesize a thioamidepeptide. GLP-1-A^(s) ₈ is exemplified in the reaction scheme.

FIG. 19 illustrates the effects of thioamide modification at scissilebond position in glucose-dependent insulinotropic peptide (GIP)(GIP-A^(s) ₄). Glucose-dependent insulinotropic peptide (GIP) stimulatespancreatic insulin secretion and fatty acid metabolism. GIP secretion isreduced in type 2 diabetes patients. Thioamide modification in GIPdramatically increase its proteolytic half-life to about 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to the use of thioamidemodifications (0-to-S substitutions of the peptide bond) in biologicallyactive peptides as a way to generate modified peptides that arestabilized against proteolytic degradation, and have approximately thesame biological activity as the parent peptide. In certain embodiments,thioamide modification at the cleavage site of a peptide decreasesproteolysis rates, in certain instances by as much as 1,000-fold, thusgreatly improving the pharmacokinetics of the peptide. In otherembodiments, thioamide modification of a peptide does not significantlyalter the structure of the peptide and does not disrupt receptor bindingor biological activity of the peptide. In yet other embodiments,thioamide modification of a peptide does not increase immune systemrecognition of the peptide as compared to the unmodified peptide.

As demonstrated herein, thioamide modification of biologically activepeptides (such as GLP-1) stabilizes the peptide towardsprotease-mediated degradation without substantially altering theirbiological activities. In certain embodiments, the thioamide modifiedGLP-1 peptides of the invention stabilize the GLP-1 peptide towardsproteolysis by proteases.

In certain embodiments, the thioamide-modified peptides of the inventionhave longer half-lives in vivo than the corresponding unmodifiedpeptides. In other embodiments, the thioamide-modified peptides of theinvention act as inhibitors of the proteases that cause proteolyticdegradation of the corresponding unmodified peptides, in the case wherethe protease hydrolyzes the peptide bond that is replaced with thethioamide in the modified peptides.

In certain embodiments, in those cases wherein the thioamidemodification is between the second and third amino acid residues fromthe N-terminus of the peptide and wherein the N-terminus residue of thepeptide is histidine, the stability of thioamide-modified peptides isfurther increased by replacing the N-terminus residue with another aminoacid, such as but not limited to, phenylalanine. In certain embodiments,the amino acid replacing the histidine does not comprises an imidazoleside chain.

In certain embodiments, the thioamide-modified peptides of the inventionare further modified, by using methods such as but not limited to:methylation of one or more NH groups in the peptide backbone; amidationand/or esterification of the C-terminus carboxyl group and/or any sidechain carboxyl group; alkylation, acylation, carbamoylation and/orsulfonylation of the N-terminus amino group and/or any side chain aminogroup; and any other peptide modification known in the art.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, specific methods andmaterials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “abnormal” when used in the context of organisms, tissues,cells or components thereof refers to those organisms, tissues, cells orcomponents thereof that differ in at least one observable or detectablecharacteristic (e.g., age, treatment, time of day, and so forth) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics that arenormal or expected for one cell or tissue type, might be abnormal for adifferent cell or tissue type.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

As used herein, the term “amino acid” refers to any natural ornon-natural compound having a carboxyl group and an amino group in amolecule. An “amino acid” as used herein is meant to include bothnatural and synthetic amino acids, and both D- and L-amino acids.“Natural amino acid” means any of the twenty L-amino acids commonlyfound in naturally occurring peptides. “Non-natural amino acid residues”means any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or derived from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and substitutions. Amino acids contained within thepeptides, and particularly at the carboxy- or amino-terminus, can bemodified by methylation, amidation, acetylation or substitution withother chemical groups which can change a peptide's circulating half-lifewithout adversely affecting activity of the peptide. Additionally, adisulfide linkage may be present or absent in the peptides.

As used herein, natural amino acids are represented by the full namethereof, by the three letter code corresponding thereto, or by theone-letter code corresponding thereto, as indicated below:

Three-Letter One-Letter Full Name Code Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

As used herein, the term “BNP” refers to brain natriuretic peptide, andcorresponds to the peptide of SEQ ID NO:7 or a salt or solvate thereof

As used herein, the term “CD” refers to circular dichroism.

As used herein, the term “DPP-4” refers to dipeptidyl peptidase-4,adenosine deaminase complexing protein 2 or CD26 (cluster ofdifferentiation 26). In certain embodiments, DPP-4 is mammalian, such ashuman.

As used herein, the term “exenatide” refers to the peptide of SEQ IDNO:3 or a salt or solvate thereof.

As used herein, the term “GIP” refers to glucose-dependentinsulinotropic factor. In certain embodiments, the GIP is mammalian,such as human. In other embodiments, GIP comprises the peptide of SEQ IDNO:5 or a salt or solvate thereof.

As used herein, the term “GLP-1” refers to glucagon-like peptide-1fragment 7-36. In certain embodiments, GLP-1 is human. In otherembodiments, GLP-1 comprises the peptide of SEQ ID NO:1 or a salt orsolvate thereof.

As used herein, the term “thio GLP-1” refers to GLP-1 wherein at leastone peptidic bond is replaced with a thioamide. In certain embodiments,thio GLP-1 comprises the peptide of SEQ ID NO:2 or a salt or solvatethereof.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression that can be usedto communicate the usefulness of a compound, composition, or method ofthe invention in the kit for effecting prevention or treatment of thevarious diseases or disorders recited herein. Optionally, oralternately, the instructional material can describe one or more methodsof preventing or treating the diseases or disorders in a cell or atissue of a mammal. The instructional material of the kit of theinvention can, for example, be affixed to a container that contains theidentified compound or composition of the invention or be shippedtogether with a container which contains the identified compound orcomposition. Alternatively, the instructional material can be shippedseparately from the container with the intention that the instructionalmaterial and the compound or composition be used cooperatively by therecipient.

A “label” or “detectable label” or “tag” is a composition detectable bymass spectrometric, spectroscopic, photochemical, biochemical,immunochemical, or chemical means. For example, useful labels includeradioactive isotopes (e.g., ³H, ³⁵S, ³²P, ⁵¹Cr or ¹²⁵I), stable isotopes(e.g., ¹³C, ¹⁵N or ¹⁸O), fluorescent dyes, electron-dense reagents,enzymes (e.g., alkaline phosphatase, horseradish peroxidase, or otherscommonly used in an ELISA), biotin, digoxigenin, or haptens or epitopesand proteins for which antisera or monoclonal antibodies are available.In general, a label as used in the context of the present invention isany entity that may be used to detect or isolate the product ofinterest. Thus, any entity that is capable of binding to another entitymay be used in the practice of this invention, including withoutlimitation, epitopes for antibodies, ligands for receptors, and nucleicacids, which may interact with a second entity through means such ascomplementary base pair hybridization.

As used herein, the term “ligation” as applied to two or more moleculesrefers to the process to creating covalent chemical bonds among the twoor more molecules, as to form at least one molecule that incorporates atleast a portion of each of the two or more molecules.

As used herein, the term “liraglutide” refers to the peptide of SEQ IDNO:4(H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(γ-Glu-palmitoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH)or a salt or solvate thereof.

As used herein, the term “MALDI MS” refers to matrix-assisted laserdesorption/ionization mass spectrometry.

As used herein, the term “OXM” refers to oxyntomodulin, corresponding tothe peptide of SEQ ID NO:6 or a salt or solvate thereof.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In a non-limiting embodiment, the patient, subject or individual is ahuman.

The terms “peptide,” “polypeptide” and “protein,” as used herein, areinterchangebly used to define as a chain of amino acid residues, usuallyhaving a defined sequence. As used herein, the term polypeptide ismutually inclusive of the terms “peptide” and “protein.” “Polypeptide”also refers to a polymer composed of amino acid residues, relatednaturally occurring structural variants, and synthetic non-naturallyoccurring analogs thereof linked via peptide bonds, related naturallyoccurring structural variants, and synthetic non-naturally occurringanalogs thereof. Synthetic polypeptides can be synthesized, for example,using an automated polypeptide synthesizer.

Conventional notation is used herein to portray polypeptide sequences:the left-hand end of a polypeptide sequence is the amino-terminus; theright-hand end of a polypeptide sequence is the carboxyl-terminus.Peptidic bonds are formed by amides (also known as oxiamides).

“Proteases” (or “proteinases”, “peptidases”, or “proteolytic” enzymes)generally refer to a class of enzymes that cleave peptide bonds betweenamino acids of proteins. Because proteases use a molecule of water toeffect hydrolysis of peptide bonds, these enzymes can also be classifiedas hydrolases. Six classes of proteases are presently known: serineproteases, threonine proteases, cysteine proteases, aspartic acidproteases, metalloproteases, and glutamic acid proteases (see, e.g.,Barrett A. J. et al., The Handbook of Proteolytic Enzymes, 2^(nd) ed.Academic Press, 2003). Proteases are involved in a multitude ofphysiological reactions from simple digestion of food proteins to highlyregulated cascades (e.g., the cell cycle, the blood clotting cascade,the complement system, and apoptosis pathways). It is well known to theskilled artisan that proteases can break either specific peptide bonds,depending on the amino acid sequence of a protein, or break down apolypeptide to the constituent amino acids.

As used herein, the term “salt” embraces addition salts of free acids orfree bases that are compounds useful within the invention. Suitable acidaddition salts may be prepared from an inorganic acid or from an organicacid. Examples of inorganic acids include hydrochloric, hydrobromic,hydriodic, nitric, carbonic, sulfuric, phosphoric acids, perchloric andtetrafluoroboronic acids. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of whichinclude formic, acetic, propionic, succinic, glycolic, gluconic, lactic,malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic,phenylacetic, mandelic, embonic (pamoic), methanesulfonic,ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid. Suitable base addition salts ofcompounds useful within the invention include, for example, metallicsalts including alkali metal, alkaline earth metal and transition metalsalts such as, for example, lithium, calcium, magnesium, potassium,sodium and zinc salts. Acceptable base addition salts also includeorganic salts made from basic amines such as, for example,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methyl-glucamine) and procaine. All ofthese salts may be prepared by conventional means from the correspondingfree base compound by reacting, for example, the appropriate acid orbase with the corresponding free base.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” or “effective amount” of a compoundis that amount of compound that is sufficient to provide a beneficialeffect to the subject to which the compound is administered. In certainembodiments, administration of a therapeutically effective amount of thecompound prevents or treats (delays or prevents the onset of, preventsthe progression of, inhibits, decreases or reverses) a disease orcondition described herein, including alleviating symptoms of suchdiseases. An “effective amount” of a delivery vehicle is that amountsufficient to effectively bind or deliver a compound.

As used herein, the term “thioamide” refers to the group —C(═S)—NR—,wherein R is selected from the group consisting of H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted cycloalkyl, optionallysubstituted cycloalkenyl, optionally substituted cycloalkynyl,optionally substituted aryl, optionally substituted heteroaryl, andoptionally substituted heterocyclyl. In certain embodiments, thioamidescorresponds to amides (or oxiamides) wherein the carbonyl group of theamide bond is replaced with a thiocarbonyl group.

As used herein, the term “thioamide-modified” or “thioamide-substituted”peptide, polypeptide or protein refers to a peptide, polypeptide orprotein wherein at least one peptide bond is replaced with a thioamidebond. In certain embodiments, the thioamide is located between thesecond and third amino acid residues from the N-terminus of the peptide,polypeptide or protein.

As used herein, an amino acid denoted as “AA*” comprises an amino acidAA that is thioamide-modified, wherein the carbonyl group of the aminoacid is replaced with a thiocarbonyl. In certain embodiments, a proteasecleaves a peptide at the dipeptide site AA1-AA2, wherein AA1 and AA2 areaminoacids. The protease cleaves the peptidic bond between AA1 and AA2to form a N-terminus peptide fragment comprising AA1 as the C-terminusresidue, and a C-terminus peptide fragment comprising AA2 as theN-terminus fragment. In other embodiments, the peptide comprising thedipeptide cleavage site AA1*-AA2 is cleaved by the protease at a lowerrate than the peptide comprising the dipeptide cleavage site AA1-AA2.

For the purpose of notation used herein, the thioamide linkage betweenamino acid AA1 and amino acid AA2, wherein the thiocarbonyl group isderived from amino acid AA1 is denoted as AA1*-AA2. In this notation,the AA1 residue constitutes the N-terminus and the AA2 residueconstitutes the C-terminus of the thioamide-modified dipeptide AA1*-AA2.

As used herein, “treating a disease or disorder” means reducing thefrequency with which a symptom of the disease or disorder is experiencedby a patient. Disease and disorder are used interchangeably herein.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

In one aspect, the present invention relates to the use of thioamidemodifications (O-to-S substitutions of the peptide bond) in biologicallyactive peptides as a way to generate thioamide-modified peptides thatare stabilized against proteolytic degradation, and have approximatelythe same biological activity as the parent peptide. In certainembodiments, thioamide modification at the cleavage site of a peptidedecreases proteolysis rates by as much as 1,000-fold, thus greatlyimproving the pharmacokinetics of the peptide. In other embodiments,thioamide modification of a peptide does not significantly alter thestructure of the peptide and does not disrupt receptor binding orbiological activity of the peptide. In yet other embodiments, thioamidemodification of a peptide does not increase immune system recognition ofthe peptide as compared to the unmodified peptide.

The peptide contemplated in the invention can be any peptide that iscapable of undergoing to proteolytic degradation. In certainembodiments, the peptide is a dipeptidyl peptidase 4 (DPP-4) substrateor a carboxypeptidase substrate. The DPP-4 substrates include, but isnot limited to, glucagon family peptides, pancreatic polypeptide familypeptides, and chemokine family peptides.

Glucagon family peptides include, but are not limited to, glucagon (SEQID NO:21), pituitary adenylate cyclase-activating peptide (PACAP) (SEQID NO:39), vasoactive intestinal peptide (VIP) (SEQ ID NO:41), GLP-1(SEQ ID NO:1), oxyntomodulin (OXM) (SEQ ID NO:6), GIP (SEQ ID NO:5),growth-hormone-releasing hormone (GHRH) (SEQ ID NO:43), sermorelin (GRF)(SEQ ID NO:45), and tesamorelin (SEQ ID NO:49).

Pancreatic polypeptide family peptides include, but are not limited to,peptide YY (PYY) (SEQ ID NO:25), pancreatic polypeptide (PP) (SEQ IDNO:27), B-type natriuretic peptide (BNP) (SEQ ID NO:7), neuropeptide Y(NPY) (SEQ ID NO:23), and enterostatin (ENT) (SEQ ID NO:47).

Chemokine family peptides include, but are not limited to, RANTES (CCL5)(SEQ ID NO:29), CCL2 (SEQ ID NO:31), CCL8 (SEQ ID NO:33), CCL7 (SEQ IDNO:35), and CCL13 (SEQ ID NO:37).

DPP-4 substrates play various important roles in regulatingphysiological and behavior activities, for example, insulin secretion(GLP-1, GIP), appetite (ENT), mood and stress (NPY), immune cellfunction (RANTES), and growth and development (GHRH). Several of thesepeptides are currently prescribed or under clinical trials in unmodifiedforms. In certain embodiments, modification with a thioamide residue atthe indicated position should slow or eliminate proteolytic degradationby DPP-4. In other embodiments, degradation by other enzymes at otherpositions may also be limiting for in vitro or in vivo stability, andthioamide substitution at those position may further stabilize thepeptide.

The hormones GIP, GLP-1, and OXM act as glucose-lowering agents and havebeen studied extensively as diabetes treatments.

Brain or B-type natriuretic peptide (BNP) is a 32-amino acid peptidesecreted by the ventricles of the heart, causing a decrease in systemicvascular resistance and central venous pressure as well as an increasein natriuresis (lowering of sodium in the blood). BNP plays an importantrole in the body's defense against hypertension and is used as atreatment of congestive heart failure.

Growth-hormone-releasing hormone (GHRH), also known as growth-hormonereleasing factor (GRF, GHRF), somatoliberin or somatocrinin, is producedin the arcuate nucleus of the hypothalamus. Semorelin is the truncatedform of GHRH and is prescribed for growth hormone deficiency inchildren. Tesamorelin has an N-terminal modification of GHRH and isprescribed for lipodystrophy in HIV patients.

Neuropeptide Y (NPY) is a 36-amino acid peptide that acts as aneurotransmitter in the brain and in the autonomic nervous system. Inthe autonomic system it serves as a strong vasoconstrictor and causesfat accumulation. In the brain, it reduces anxiety and stress, as wellas perception of pain.

Also, various DPP-4 substrates have short half-lives. For example,GLP-1, GIP, BNP, and OXM have in vivo half-lives of less than 15minutes. NPY has a half-life of 15-20 minutes. ENT has a half-life of 5minutes. GHRH has a half-life of 19 minutes.

DPP-4 inhibition affects the levels of these peptides in circulation aswell as GLP-1. On the other hand, stabilized versions of GIP, OXM,GLP-1, NPY, GHRH, ENT, or BNP should act more selectively than DPP-4inhibition, by impacting only one signaling pathway. In certainembodiments, the invention provides a thioamide-modified analog of apeptide, wherein the thioamide modification occurs at the peptidic bondthat is cleaved by a protease. In other embodiments, the proteasecomprises DPP-4. In yet other embodiments, the protease comprisescarboxypeptidase. In yet other embodiments, the peptide comprises aDPP-4 substrate. In certain instances, the peptide comprises GIP, OXM,GLP-1, NPY, GHRH, ENT, or BNP. In yet other embodiments, the thioamidemodification reduces the rate at which the peptide is cleaved by theprotease. In yet other embodiments, the peptide bond that is cleaved bythe protease is subject to the thioamide modification. In yet otherembodiments, a peptide bond that is neighboring to the peptide bondcleaved by the protease is subject to the thioamide modification.

As demonstrated herein in a non-limiting manner, the invention may beimplemented using incretin hormone glucagon-like peptide 1 (GLP-1),which stimulates insulin and suppresses glucagon secretion, inhibitsgastric emptying, and reduces appetite and food intake. GLP-1 isinactivated through proteolytic cleavage by dipeptidyl peptidase 4(DPP-4) with a half-life of less than 2 minutes. Stabilized GLP-1analogs exenatide (BYETTA®) and liraglutide (VICTOZA®) are currentlyprescribed as Type 2 diabetes drugs.

As demonstrated herein, thioamide modification at the scissile bond ofGLP-1 (thio GLP-1, X═S in FIG. 1, SEQ ID NO:2, wherein Xaa (1) ishistidine) increased the GLP-1 half-life to ranges (>24 hours)comparable to exenatide and liraglutide. For this study, purifiedpeptides were incubated with DPP-4 in assay buffer for various timeperiods. The reactions were quenched, and the products analyzed byHPLC/MALDI MS to determine the amount of intact peptide. The degradationdata indicated that the half-life of thio GLP-1 is greater than about 24hours under conditions where the half-life of GLP-1 is 21 minutes. Thus,an at least 100-fold increase in stability was obtained by introducing asingle atom substitution in GLP-1.

To further investigate the interaction between DPP-4 and thio GLP-1,kinetic assays showed that thio GLP-1 acts as a competitive DPP-4inhibitor. Thus, thio GLP-1 acts as both a stabilized GLP-1R agonist anda DPP-4 inhibitor. In certain embodiments, this unique attribute affectsthe in vivo pharmacology of thio GLP-1.

The effects of thioamide modification on the structure of GLP-1 werealso studied. CD spectroscopy was used to examine the secondarystructure of GLP-1 and thio GLP-1 in the far UV region, showing that thethioamide at Ala₈ was not disruptive to GLP-1 folding, since the CDsignatures of thio GLP-1 and GLP-1 were identical.

Further, the activity of thio GLP-1 as compared to GLP-1 was tested invivo using intra-peritoneal glucose tolerance tests (IPGTTs). In theseexperiments, mice were injected with the peptide 60 minutes prior toadministration of a glucose challenge (time=0). Blood glucose levelswere measured at −90, 0, 30, 60, 90 and 120 minutes. Thio GLP-1 greatlyimproved glucose tolerance and led to significantly lower blood glucoselevels at every time point, to a greater degree than GLP-1. These datademonstrate that thio GLP-1 is active in mice.

As demonstrated herein in a non-limiting manner, the invention may alsobe implemented using glucose-dependent insulinotropic peptide (GIP) (SEQID NO:5). GIP stimulates pancreatic insulin secretion and fatty acidmetabolism. GIP secretion is reduced in type 2 diabetes patients.Thioamide modification at the scissile position in GIP (GIP-A^(S) ₄, SEQID NO:13) increase its proteolytic half-life dramatically to about 24hours (FIG. 19).

Although the embodiments have demonstrated thioamide modification at thescissile bond of a peptide (such as a DPP-4 substrate), one of ordinaryskill in the art would appreciate the thioamide modification can be atany position of the peptide under consideration. Thioamide modificationat a different position from the scissile position in the DPP-4substrate (or any other position thought to allow the peptide to bedegraded by protease) may have distinct therapeutic effects. As such,thioamide modification at any possible position of the DPP-4 substrateis contemplated within the present invention.

It is also contemplated within the invention to have two or morethioamide modifications in a peptide. Such multiple thioamidemodification can help stabilize the peptide against proteolyticdegradation caused by DPP-4, as well as other proteases. Onenon-limiting example is enterostatin (ENT), expressed as a precursorprotein in the pancreas, stomach, duodenal mucosa, and specific brainregions. ENT is a peptide having the sequence of AP₂GP₄R (the subscriptsare used to differentiate the positions of two Ps only), and selectivelyinhibits the intake of dietary fat in rodent models given a choice ofdiets. ENT is subject to DPP-4 proteolysis and carboxypeptidase at theP₂ and P₄ positions respectively. Accordingly, thioamide modificationsat P₂ and P₄ positions make ENT more stable in the presence of DPP-4 andcarboxypeptidase.

Compositions

The invention includes a thioamide-substituted peptide, wherein thepeptide is modified with a thioamide at the peptidic bond that iscleaved by a protease.

In certain embodiments, the peptide comprises a peptide selected fromthe group consisting of:

SEQ ID NO: 2, wherein Xaa (1) is histidine(thio GLP-1):HA*EGTFTSDVSSYLEGQAAKEFIAWLVKGR;SEQ ID NO: 2, wherein Xaa (1) is phenylalanine (GLP-1-F⁷AS₈):FA*EGTFTSDVSSYLEGQAAKEFIAWLVKGR;SEQ ID NO: 11, wherein Xaa (1) is histidine (thio exenatide):HG*EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPS;SEQ ID NO: 11, wherein Xaa (1) is phenylalanine:FG*EGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPS;SEQ ID NO: 12, wherein Xaa (1) is histidine(thio liraglutide):HA*EGTFTSDVSSYLEGQAAXEFIAWLVRGRG, [X = Lys(γ-Glu-palmitoyl)];SEQ ID NO: 12, wherein Xaa (1) is phenylalanine:FA*EGTFTSDVSSYLEGQAAXEFIAWLVRGRG, [X = Lys(γ-Glu-palmitoyl)];SEQ ID NO: 13 (thio GIP): YA*EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ;SEQ ID NO: 14, wherein Xaa (1) is histidine (thio OXM):HS*QGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA;SEQ ID NO: 14, wherein Xaa (1) is phenylalanine:FS*QGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA; SEQ ID NO: 15 (thio BNP):SP*KMVQGSGCFGRKMDRISSSSGLGCKVLRRH;SEQ ID NO: 16, wherein Xaa (1) is histidine (thio M1):Hα*EGTFTSDVSSYLEGQAAKEFIAWLVKαR, (α = dimethyl Gly);SEQ ID NO: 16, wherein Xaa (1) is phenylalanine:Fα*EGTFTSDVSSYLEGQAAKEFIAWLVKαR, (α = dimethyl Gly);SEQ ID NO: 17, wherein Xaa (1) is histidine (thio M2):HA*αGTFTSDVSSYLEGQAAKEFIAWLVKGR, (α = t-butyl Gly);SEQ ID NO: 17, wherein Xaa (1) phenylalanine:FA*αGTFTSDVSSYLEGQAAKEFIAWLVKGR, (α = t-butyl Gly);SEQ ID NO: 18, wherein Xaa (1) is histidine (thio M3):HA*αGTFTSDVSSYLEGQAAKEFIAWLVKGR, (α = β,β-D);SEQ ID NO: 18, wherein Xaa (1) is phenylalanine:FA*αGTFTSDVSSYLEGQAAKEFIAWLVKGR, (α = β,β-D);SEQ ID NO: 22, wherein Xaa (1) is histidine (thio Glucagon):HS*QGTFTSDYSKYLDSRRAQDFVQWLMNT;SEQ ID NO: 22, wherein Xaa (1) is phenylalanine:FS*QGTFTSDYSKYLDSRRAQDFVQWLMNT; SEQ ID NO: 24 (thio NPY):YP*SKPDNPGEDAPAEDMARYYSALRHYINLITRQRY; SEQ ID NO: 26 (thio PYY):YP*IKPEAPGEDASPEELNRYYASLRHYINLITRQRY;SEQ ID NO: 28 (thio pancreatic polypeptide (PP)):AP*LEPVYPGDNATPEQMAQYAADLRRYINNILTRPRY;SEQ ID NO: 30 (thio RANTES (CCL5)):SP*YSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEMS;SEQ ID NO: 32 (thio CCL2):QP*DAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLD;SEQ ID NO: 34 (thio CCL8):QP*DSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLKP;SEQ ID NO: 36 (thio CCL7):QP*VGINTSTTCCYRFINRKIPKQRLESYRRTTSSHCPKEAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL;SEQ ID NO: 38 (thio CCL13):QP*DALNAPVTCCFTFSSRKISLQRLKSYVITTSRCPQKAVIFRTKLGKEICADPKEKWVQNYMKHLGRKAHTLKT;SEQ ID NO: 40, wherein Xaa (1) is histidine(thio PACAP):HS*DGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK;SEQ ID NO: 40, Xaa (1) is phenylalanine:FS*DGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK;SEQ ID NO: 42, wherein Xaa (1) is histidine (thio vasoactive intestinal peptide (VIP)):HS*DAVFTDNYTRLRKQMAVKKYLNSILN;SEQ ID NO: 42, wherein Xaa (1) is phenylalanine:FS*DAVFTDNYTRLRKQMAVKKYLNSILN; SEQ ID NO: 44 (thio GHRH):YA*DAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL; SEQ ID NO: 46 (thio GRF):YA*DAIFTNSYRKVLGQLSARKLLQDIMSR; SEQ ID NO: 48 (thio ENT): AP*GPR;SEQ ID NO: 50 (thio Tesamorelin):Y^(#)A*DAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL;SEQ ID NO: 51 (bithio ENT) AP*GP*R;wherein the C-terminus is optionally amide protected, and the amino acidmarked as * is thioamide-modified; a salt or solvate thereof, and anycombinations thereof.

The invention further comprises a pharmaceutical composition comprisingat least one peptide of the invention and a pharmaceutically acceptablecarrier.

Methods

The invention includes a method of stabilizing a peptide againstprotease hydrolysis, the method comprising modifying with a thioamidethe peptidic bond that the protease hydrolyzes. In certain embodiments,the protease comprises DPP-4. In other embodiments, the thioamide isformed between the second and third amino acid residues from theN-terminus of the peptide. In yet other embodiments, the peptidecomprises a DPP-4 substrate. In certain instances, the peptide comprisesGLP-1, GIP, OXM, BNP, ENT, GHRH, NPY or any combinations thereof. In yetother embodiments, the thioamide-modified peptide has equivalentbiological activity to the peptide. In yet other embodiments, thethioamide-modified peptide has longer in vivo half-life than thepeptide.

The invention further includes a method of treating or preventingdiabetes or obesity in a subject in need thereof, wherein the methodcomprises administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising a thioamide-modifiedpeptide. In certain embodiments, the thioamide-modified peptidecomprises at least one selected from the group consisting of SEQ IDNOs:2, 11-14, 16-18, 20, 22, 48 51 or a salt or solvate thereof. Inother embodiments, the thioamide-modified peptide has at least oneeffect selected from the group consisting of stimulate insulinproduction in the subject, suppress glucagon secretion in the subject,inhibit gastric emptying in the subject, reduce appetite in the subject,and reduce food intake in the subject.

The invention further includes a method of treating or preventing acardiac disease or disorder in a subject in need thereof, wherein themethod comprises administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising athioamide-modified peptide. In certain embodiments, thethioamide-modified peptide comprises SEQ ID NO:15, or a salt or solvatethereof. In other embodiments, the cardiac disease or disorder compriseshypertension or congestive heart failure.

Kits

The invention includes a kit comprising a thioamide-modified peptide ofthe invention, and an instructional material for use thereof. Theinstructional material comprises instructions for treating or preventinga disease or disorder in a subject using the thioamide-modified peptideof the invention. In certain embodiments, the kit further comprises atleast one additional agent that treats or prevents the disease ordisorder in the subject.

Combination Therapies

In certain embodiments, the compounds of the invention are useful in themethods of the invention in combination with at least one additionalcompound useful for treating or preventing a disease or disordercontemplated within the invention. This additional compound may comprisecompounds identified herein or compounds, e.g., commercially availablecompounds, known to treat, prevent or reduce the symptoms of a diseaseor disorder contemplated within the invention.

For treatment or prevention of diabetes or obesity, the additionalcompound may be selected from the group consisting of GLP-1; GIP; OXM;meglitinides, such as repaglinide (PRANDIN®), and nateglinide(STARLIX®); sulfonylureas, such as glipizide (GLUCOTROL®), glimepiride(AMARYL®), and glyburide (DIABETA®, GLYNASE®); DPP-4 inhibitors, such assaxagliptin (ONGLYZA®), sitagliptin (JANUVIA®), and linagliptin(TRADJENTA®); biguanides, such as metformin (FORTAMET®, GLUCOPHAGE®, andothers); thiazolidinediones, such as rosiglitazone (AVANDIA®) andpioglitazone (ACTOS®); alpha-glucosidase inhibitors, such as acarbose(PRECOSE®) and miglitol (GLYSET®); amylin mimetics, such as pramlintide(SYMLIN®); incretin mimetics, such as exenatide (BYETTA®) andliraglutide (VICTOZA®); insulin and insulin analogs and derivatives.

For treatment or prevention of a cardiac disorder or disease, theadditional compound may be selected from the group consisting ofaspirin, statins, thiazide-based diuretics, angiotensin convertingenzyme inhibitors, angiotensin II receptor blockers, calcium channelblockers, vasodilators, aldosterone receptor antagonists, beta-blockers,and alpha-blockers.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 19981, Clin. Pharmacokinet. 6: 429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22:27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the onset of a disease or disorder contemplatedin the invention. Further, several divided dosages, as well as staggereddosages may be administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection. Further, the dosagesof the therapeutic formulations may be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions of the present invention to a patientor subject, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder contemplated in the invention. Aneffective amount of the therapeutic compound necessary to achieve atherapeutic effect may vary according to factors such as the state ofthe disease or disorder in the patient; the age, sex, and weight of thepatient; and the ability of the therapeutic compound to treat a diseaseor disorder contemplated in the invention. Dosage regimens may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation. A non-limiting example of an effective dose range for atherapeutic compound of the invention is from about 1 and 5,000 mg/kg ofbody weight/per day. One of ordinary skill in the art would be able tostudy the relevant factors and make the determination regarding theeffective amount of the therapeutic compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease or disorder contemplated in the invention.

In certain embodiments, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Incertain embodiments, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

In certain embodiments, the compositions of the invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of the inventionare administered to the patient in range of dosages that include, butare not limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from individual toindividual depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient is determined by the attending physicaltaking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof a disease or disorder contemplated in the invention.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude, but are not limited to, nasal, inhalational, topical, oral,buccal, rectal, pleural, peritoneal, vaginal, intramuscular,subcutaneous, transdermal, epidural, intratracheal, otic, intraocular,intrathecal or intravenous route. The compounds for use in the inventionmay be formulated for administration by any suitable route, such as fororal or parenteral, for example, transdermal, transmucosal (e.g.,sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g.,trans- and perivaginally), (intra)nasal and (trans)rectal),intravesical, intrapulmonary, intraduodenal, intragastrical,intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial,intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compounds of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of a disease or disorder contemplated in theinvention. Using a wax/pH-sensitive polymer mix, a gastric insolublecomposition may be obtained in which the active ingredient is entrapped,ensuring its delayed release.

Parenteral Administration

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intravenous, intraperitoneal, intramuscular, intrasternal injection, andkidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multidose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer system. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.2003/0147952; 2003/0104062; 2003/0104053; 2003/0044466; 2003/0039688;and 2002/0051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In one embodiment of the invention, the compounds of the invention areadministered to a patient, alone or in combination with anotherpharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that may,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of thepresent invention depends on the age, sex and weight of the patient, thecurrent medical condition of the patient and the progression of adisease or disorder contemplated in the invention. The skilled artisanis able to determine appropriate dosages depending on these and otherfactors.

A suitable dose of a compound of the present invention may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of thedisease or disorder, to a level at which the improved disease isretained. In certain embodiments, patients require intermittenttreatment on a long-term basis upon any recurrence of symptoms and/orinfection.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withminimal toxicity. The dosage optionally varies within this rangedepending upon the dosage form employed and the route of administrationutilized.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations that are evident as a result of the teachings providedherein.

Materials and Methods

In these studies Mus musculus; lean and diet-induced obese, DIO,C57BL6/J mice are used. In certain embodiments, mice may be used tostudy the ability of the peptides of the invention to regulate bloodglucose levels during a glucose tolerance test. Mice are injectedintraperitoneally using 28 gauge insulin syringes with the peptidesdissolved in saline, administered in a volume corresponding to 0.5% ofbody mass. Mice are monitored following compound administration for anysigns of adverse reactions.

Thirty minutes after injection with the peptides, mice are injectedintraperitoneally with a sterile solution of 20% D-glucose in saline(1-2 g/kg) using a 28 gauge insulin needle. Administered volume of thesaline glucose solution does not exceed 1% of body mass (e.g., 0.3 mLfor a 30 g mouse). Blood glucose measurements is performed just prior toinjection of the peptide (−30 minutes), before glucose injection (0minutes), and at 15, 30, 60, and 120 minutes after injection. Over thecourse of the 150 minute experiment, a total of 30 μL (5 μL per timepoint) of blood are collected. At the end of the measurement period,mice are sacrificed by CO₂ inhalation, and blood is collected to measureresidual peptide levels. In addition, a similar set of experiments isperformed, wherein the peptides are injected 300-360 minutes prior toglucose injection to determine whether thio peptide has a longerhalf-life than the parent peptide.

Male mice that are at least 16 weeks, but younger than 24 weeks old, areused. For each condition (thio peptide, peptide, and vehicle), 8 miceare used, for a total of 24 mice per experiment.

Example 1: Selected Thioamide Studies

Thioamides can quench fluorophores such as methoxycoumarin (Mcm, μ), andthis quenching can be used to monitor protein folding and stability(Goldberg, et al., 2014, J. Am. Chem. Soc. 135:2086-2093). Thioamidesmay be incorporated into peptides made on solid phase usingbenzotriazole precursors like the thioleucine precursor shown in FIG. 2.Thioamide-containing peptides of up to 35 residues can be synthesized inreasonable yields (˜50% of the corresponding oxoamide peptide), makingsynthesis of any of the thioamide peptide hormones considered hereinstraightforward (Goldberg, et al., 2010, J. Am. Chem. Soc.132:14718-14720). Thioamides can be used in fluorogenic constructs tomonitor proteolysis by relief of a quenching interaction (FIG. 2).Cleavage of the substrate L'LKAAμ by papain is shown in FIG. 2. Nochange in fluorescence was seen for the oxoamide (LLKAμ), but it wasproteolyzed at the same rate as the thiopeptide in an HPLC assay. Thethioamide was not disruptive to proteolysis when placed at a site two orthree residues away from the scissile bond. Taken together with thestudies of thioamide suppression of proteolysis when placed directly atthe scissile bond, the data indicate that the effect of the thioamide isvery local. Additionally, studies of the stability of thioamides in celllysates using non-proteolyzable D-amino acid thiopeptides indicated thatthey are not substantially metabolized by other enzymes.

Example 2: In Vivo GLP-1 Activity Assays

Physiological assays are used to assess the activity of thioamidevariants of GLP-1 in regulating glucose control. The activity andhalf-lives of GLP-1 and exenatide may be determined using glucosetolerance tests (GTTs). In these experiments, mice are injectedintraperitoneally (i.p.) with the peptide 60 minutes prior toadministration of a glucose challenge (time=0 minutes). Blood glucoselevels are measured at −60, 0, 30, 60, 90 and 120 minutes. GLP-1 andexenatide greatly improved glucose tolerance and led to significantlylower blood glucose levels at every time point (FIG. 3, left). Thisdemonstrate that GTTs may be used to measure the in vivo activity ofGLP-1 peptides, as well as thioamide GLP-1.

In addition, a GTT may be used to measure changes in the half-life ofthe bioactive peptides, by increasing the time between the injection ofthe peptide and the injection of glucose. In an experiment, GLP-1 orexenatide was injected, and then after 5.5 hours glucose was injected.During this waiting period GLP-1 underwent proteolysis and wasinactivated, while exenatide retained its activity. In this delayedprotocol, measurement of blood glucose levels 30 minutes after glucoseinjection showed much lower blood glucose levels in exenatide-treatedmice (p-value<0.0001), as expected (FIG. 3, right). GLP-1-treated miceexperienced only a slight, statistically-insignificant (p-value=0.13)lowering of blood glucose levels compared to vehicle-treated mice.Similar delayed GTT assays are used to determine whetherthioamide-modified GLP-1 has increased stability in vivo.

Example 3: Metabolic Stability of Thioamides

In order to shed light on the pharmaceutical applications ofthiopeptides, the stability of peptidyl thioamides towardnon-proteolytic degradation is examined. Degradation kinetics ofthiopeptides may be tested in PBS buffer, cell lysates, or mouse serum.As a way to develop a stability assay that can be used in cells, testsmay be run using a cleavable biotin tag (Yang, et al., 2010, Chem. Biol.17:1212-1222) to recover the test peptides from the mixture (FIG. 4).The test thiopeptide is labeled with an alkyne group, which reactsselectively with the azido-diazo-biotin (ADB) tag through a coppercatalyzed “click” reaction. After the appropriate incubation period, theADB-tagged samples are purified on neutravadin resin and eluted withNa₂S₂O₄. The eluent is then injected onto the HPLC for analysis. Thecorresponding alkyne-labelled peptide is synthesized for comparison.

Cell-penetrating versions of thioamides may be prepared by appending apoly-Arg sequence. Versions of these peptides are generated withfluorophores such as Acd to monitor cellular uptake and localization(FIG. 4). In addition, changes in fluorescence lifetime may be used insitu to track modifications of the thioamide that would affect quenchingof the fluorophore. Changes in lifetime may be monitored in living cellsusing fluorescence lifetime imaging microscopy (FLIM). These peptidesare incubated with MEF and HeLa cells and peptide localization analyzedby microscopy. After appropriate time points, cells are lysed andpull-down assays are performed with ADB to analyze potential thioamidemetabolites by HPLC/MS.

Example 4: Proteolysis of GLP-1 and GLP-1 Analogs

As a step in the investigation, GLP-1 itself is studied. To stabilizeGLP-1, a thioamide bond can be placed between Ala₈ and Glu₉, which isthe scissile bond of GLP 17-36 (“GLP-1”). In certain embodiments, GLP19-36 was synthesized on a microwave peptide synthesizer, and thencoupled with a thioalanine precursor and His manually. The oxoamideversion of GLP-1 was synthesized to serve as a control. Both peptideswere synthesized, purified by HPLC, and characterized by MALDI MS.

Example 5: Thio GLP-1 In Vitro Experiments

In certain embodiments, three assays are used to test the stability ofthio GLP-1 relative to GLP-1. First, in vitro degradation assays areperformed to determine the stability of thio and oxo GLP-1 in thepresence of DPP-4. Purified peptides are incubated with DPP-4 in assaybuffer for various time periods. The reaction is quenched and theproducts are analyzed by HPLC to determine the amount of intact peptide.MALDI MS is used to determine the identities of HPLC peaks.

The degradation data indicate that the half-life of thio GLP-1 isgreater than 24 hours under conditions where the half-life of GLP-1 is21 minutes. As demonstrated in FIG. 5, single atom substitution in GLP-1conferred a roughly 100-fold increase in stability. In addition to HPLCand MS assays, the hydrolysis process can also be monitored bytime-resolved UV spectroscopy. Intact thiopeptides exhibitcharacteristic UV absorption spectra with λ_(max) at 267 nm, while theλ_(max) of hydrolyzed product thioacid is at 251 nm (this peakdisappears when the thioacid further decomposes to the carboxylic acidand H₂S). Therefore, the change in absorbance of the reaction at 267 nmor 251 nm may reflect the loss of substrate. These assays may beperformed using the Tecan Infinite® M1000 plate reader. The ability tomonitor formation of the thioacid cleavage product in real time aims inunderstanding the mechanism of the proteolysis reaction. In certainembodiments, this information helps understand how thioamidemodification may be applied generally to stabilize peptides.

Cleavage of the thioamide bond may be monitored by time-resolvedfluorescence spectroscopy. The thioalanine residue can quench theintrinsic fluorescence of Trp₃₁ and Tyr₁₉ in thio GLP-1. Cleavage ofthis bond may relieve quenching by PET in a manner similar to thedesigned fluorogenic probes in the data in FIG. 2. In this case, thethioamide is placed at the scissile bond. Comparison of the fluorescenceand absorbance experiments further improves the understanding ofthioamide cleavage.

To further describe the interaction between DPP-4 and thio GLP-1,kinetic assays are performed to determine whether thio GLP-1 acts as acompetitive inhibitor of DPP-4. Various concentrations of thio GLP-1 aremixed with the same concentration of H-Ala-Pro-pNA, a commercialchromogenic substrate of DPP-4. After addition of catalytic amounts ofDPP-4, the reaction may be monitored using the M1000 plate reader. Byplotting the relationship between V₀ and the concentration of thio GLP-1in a Lineweaver-Burk analysis, the mechanism of thio GLP-1 inhibitionmay be determined. Preliminary data indicate that thio GLP-1 acts as aninhibitor, and has the unique attribute of simultaneously acting as botha stabilized GLP-1R agonist and a DPP-4 inhibitor.

In certain embodiments, thioamide modification could possibly affect thestructure or activity of GLP-1. Circular dichroism (CD) spectroscopy canbe used to examine the secondary structure of thio and oxo GLP-1 in thefar UV region. As illustrated in FIG. 5, right, the thioamide at Ala₈ isnot disruptive to GLP-1 folding as the CD signatures are nearlyidentical, with the notable exception that a small band attributable tothe thioamide can be seen at 270 nm in the thio GLP-1 spectrum. Theaffinity and agonist activity of thio and oxo GLP-1 at the human GLP-1Rare determined separately to understand the impact of thioamidemodification at Ala₈. These two properties are often uncoupled, as in apartial agonist or competitive inhibitor. Affinity is measured usingcompetition assays with fluorophore-labeled GLP-1. Activity is measuredusing cAMP Hunter eXpress GPCR assay kits (DiscoveRx). Both assays maybe performed using the M1000 plate reader.

Example 6: Thio GLP-1 Cellular Experiments

The insulin secretion activity of thio GLP-1 is measured directly usinga GSIS assay (Tinoco, et al., 2011, Biochemistry 50:2213-2222). Insulinrelease in primary mouse islets is examined using the batch releasemethod. GLP-1 or thio GLP-1 is added at 100 nM final concentration tobuffer containing basal (1.67 mM) or stimulatory (16.7 mM) glucoseconcentrations. Islets are incubated for one hour in the assay buffer at37° C. The supernatant is then collected for insulin measurement usingan ELISA, calibrated with a standard curve. In certain embodiments,these assays indicate whether (1) thio GLP-1 affects GSIS, and (2) howthis activity compares to GLP-1.

Example 7: Thio GLP-1 In Vivo Experiments

Bioactivity of the thioamide-modified peptides of the invention isevaluated in mice. In a non-limiting example, thio GLP-1 is tested inmice to determine whether it can reduce blood glucose levels after aGTT. GTTs are performed using 12-20 week-old mice, C57BL6/J wild typeand Diet-Induced Obese (DIO) mice, after a 14-hour overnight fast. DIOmice are diabetic mouse models that mimic human diabetes associated withweight gain, and experiments with these animals test the ability of thioGLP-1 to improve glucose tolerance in the context of a disease model.Mice (N=6-8 per group) are then injected i.p. with thio GLP-1, GLP-1 orexenatide (positive controls), or vehicle (negative control). Bloodglucose levels are measured at −60, 0, 15, 30, 60, and 120 minutesrelative to glucose injection.

To determine whether thio GLP-1 has a longer in vivo half-life thanGLP-1, this experiment may be repeated with the modification wherein a5-6 hour delay is inserted between the injection time and the start ofthe GTT. These animals are sacrificed at the end of the experiment, andserum levels of thio GLP-1 or GLP-1 are measured by LC-MS (Kim, et al.,2012, Proc. Natl. Acad. Sci. USA 109:8523-8527).

Example 8: Thioamide Modification in GLP-1 Analogs

Studies are performed to determine whether thioamide modification canact synergistically with other modifications to stabilize GLP-1. ThioLira, thio M1, and thio M3 are prepared as well as their oxamidecounterparts (Table 1 and FIG. 1). For these syntheses, in certainembodiments, new thioamide precursors such as the Fmoc-benzotriazoleversion of a may be prepared. The same battery of in vitro testsdescribed for example in Examples 4-6 may be applied to thesethiopeptides to determine which peptides to take forward to tests inanimals.

Example 9: Additional Signaling Peptides

In addition to GLP-1, several other peptide hormones, including GIP, OXMand BNP, play important roles in diabetes or heart health. Thioamideversions of these peptides are synthesized and then evaluated using thein vitro stability, folding, and activity assays described elsewhereherein. To synthesize these peptides, new thioamide precursors such asFmoc-thio-Ser(tBu)-benzotriazole and Fmoc-thio-Pro-benzotriazole may begenerated. Thiopeptides are tested in the DPP-4 degradation assay todetermine whether the stabilization of thioamide modification could bebroadly applied. Thio GIP may also be tested in GSIS assays to determinewhether it can retain its biological function. GLP-1R and GIP or GCGreceptor assays are performed to test the function of thio GIP and thioOXM. Thio BNP activation of natriuretic peptide receptor A may beperformed using ELISA kits. Thiopeptides may be further performed invivo assays.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. A method of stabilizing a bioactive peptideagainst protease hydrolysis, the method comprising replacing the amidebond formed between the first and second amino acid residues from theN-terminus of the bioactive peptide with a thioamide bond, wherein theprotease comprises at least one selected from the group consisting ofDPP-4 and carboxypeptidase.
 2. The method of claim 1, wherein thebioactive peptide comprises at least one selected from the groupconsisting of GIP, OCM, GLP-1, NPY, GHRH, ENT, and BNP.
 3. The method ofclaim 1, wherein the thioamide-modified peptide has equivalentbiological activity or longer in vivo half-life to the bioactivepeptide.